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The FEBS Journal Sep 2010Cytosolic sulfotransferase (SULT) SULT2B1b had previously been characterized as a cholesterol sulfotransferase. Like human SULT2B1, mouse SULT2B1b contains a unique, 31...
Cytosolic sulfotransferase (SULT) SULT2B1b had previously been characterized as a cholesterol sulfotransferase. Like human SULT2B1, mouse SULT2B1b contains a unique, 31 amino acid C-terminal sequence with a proline/serine-rich region, which is not found in members of other SULT families. To gain insight into the functional relevance of this proline/serine-rich region, we constructed a truncated mouse SULT2B1b lacking the 31 C-terminal amino acids, and compared it with the wild-type enzyme. Enzymatic characterization indicated that the catalytic activity was not significantly affected by the absence of those C-terminal residues. Glutathione S-transferase pulldown assays showed that several proteins interacted with mouse SULT2B1b specifically through this C-terminal proline/serine-rich region. Peptide mass fingerprinting revealed that of the five SULT2B1b-binding proteins analyzed, three were cytoskeletal proteins and two were cytoskeleton-binding molecular chaperones. Furthermore, wild-type mouse SULT2B1b, but not the truncated enzyme, was associated with the cytoskeleton in experiments with a cytoskeleton-stabilizing buffer. Collectively, these results suggested that the unique, extended proline/serine-rich C-terminus of mouse SULT2B1b is important for its interaction with cytoskeletal proteins. Such an interaction may allow the enzyme to move along microfilaments such as actin filaments, and catalyze the sulfation of hydroxysteroids, such as cholesterol and pregnenolone, at specific intracellular locations.
Topics: Amino Acid Sequence; Animals; Cytoskeletal Proteins; Cytoskeleton; Mice; Molecular Chaperones; NIH 3T3 Cells; Peptide Mapping; Proline; Protein Binding; Protein Interaction Domains and Motifs; Recombinant Proteins; Sequence Alignment; Serine; Sulfotransferases
PubMed: 20718863
DOI: 10.1111/j.1742-4658.2010.07781.x -
Protein Expression and Purification Oct 1992A nucleotide sequence that had been proposed for, but not identified as, rat liver aryl sulfotransferase (EC 2.8.2.1) was prepared in an appropriate vector and...
A nucleotide sequence that had been proposed for, but not identified as, rat liver aryl sulfotransferase (EC 2.8.2.1) was prepared in an appropriate vector and transformed into Escherichia coli. The protein, expressed in large amounts, was not aryl sulfotransferase (EC 2.8.2.1) but rather tyrosine-ester sulfotransferase (EC 2.8.2.9), a sulfotransferase also active with phenols but having a much wider substrate range that includes hydroxylamines and esters of tyrosine. The recombinant tyrosine-ester sulfotransferase was identified by its unique substrate spectrum, by comparison with three peptides that were sequenced from homogeneous tyrosine-ester sulfotransferase isolated directly from rat liver, and by the specificity of antibody raised to the rat liver enzyme. Two isoforms were obtained, each of which was difficult to solubilize upon sonication of E. coli. Both forms were solubilized with a solution of polyols (glycerol and sucrose) and subsequently purified to homogeneity.
Topics: Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; Escherichia coli; Genetic Vectors; Liver; Molecular Sequence Data; Plasmids; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Sulfotransferases
PubMed: 1458056
DOI: 10.1016/s1046-5928(05)80045-2 -
Trends in Biochemical Sciences Apr 1998
Topics: Amino Acid Sequence; Conserved Sequence; Models, Molecular; Molecular Sequence Data; Protein Structure, Secondary; Sequence Homology, Amino Acid; Sulfotransferases
PubMed: 9584614
DOI: 10.1016/s0968-0004(98)01182-7 -
Journal of Neurochemistry Nov 2003Sulfotransferase (SULT) 1A3 catalyzes the sulfate conjugation of catecholamines and structurally related drugs. As a step toward studies of the possible contribution of...
Sulfotransferase (SULT) 1A3 catalyzes the sulfate conjugation of catecholamines and structurally related drugs. As a step toward studies of the possible contribution of inherited variation in SULT1A3 to the pathophysiology of human disease and/or variation in response to drugs related to catecholamines, we have resequenced all seven coding exons, three upstream non-coding exons, exon-intron splice junctions and the 5'-flanking region of SULT1A3 using DNA samples from 60 African-American (AA) and 60 Caucasian-American (CA) subjects. Eight single nucleotide polymorphisms (SNPs) were observed in AA and five in CA subjects, including one non-synonymous cSNP (Lys234Asn) that was observed only in AA subjects with an allele frequency of 4.2%. This change in amino acid sequence resulted in only 28 +/- 4.5% (mean +/- SEM) of the enzyme activity of the wild-type (WT) sequence after transient expression in COS-1 cells, with a parallel decrease (54 +/- 2.2% of WT) in level of SULT1A3 immunoreactive protein. Substrate kinetic studies failed to show significant differences in apparent Km values of the two allozymes for either dopamine (10.5 versus 10.2 micro m for WT and variant, respectively) or the cosubstrate 3'-phosphoadenosine 5'-phosphosulfate (0.114 versus 0.122 micro m, respectively). The decrease in level of immunoreactive protein in response to this single change in amino acid sequence was due, at least in part, to accelerated SULT1A3 degradation through a proteasome-mediated process. These observations raise the possibility of ethnic-specific inherited alterations in catecholamine sulfation in humans.
Topics: Black or African American; Animals; Arylsulfotransferase; Base Sequence; COS Cells; Catecholamines; Cell-Free System; Crystallography, X-Ray; Enzyme Activation; Gene Expression; Gene Frequency; Genetic Linkage; Haplotypes; Humans; Introns; Molecular Sequence Data; Polymorphism, Genetic; Sequence Analysis, DNA; Sulfotransferases; White People
PubMed: 14622112
DOI: 10.1046/j.1471-4159.2003.02027.x -
Biochemical and Biophysical Research... Jun 2024Microorganisms synthesize a plethora of complex secondary metabolites, many of which are beneficial to human health, such as anticancer agents and antibiotics. Among...
Microorganisms synthesize a plethora of complex secondary metabolites, many of which are beneficial to human health, such as anticancer agents and antibiotics. Among these, the Sungeidines are a distinct class of secondary metabolites known for their bulky and intricate structures. They are produced by a specific biosynthetic gene cluster within the genome of the soil-dwelling actinomycete Micromonospora sp. MD118. A notable enzyme in the Sungeidine biosynthetic pathway is the activating sulfotransferase SgdX2. In this pathway, SgdX2 mediates a key sulfation step, after which the product undergoes spontaneous dehydration to yield a Sungeidine compound. To delineate the structural basis for SgdX2's substrate recognition and catalytic action, we have determined the crystal structure of SgdX2 in complex with its sulfate donor product, 3'-phosphoadenosine 5'-phosphate (PAP), at a resolution of 1.6 Å. Although SgdX2 presents a compact overall structure, its core elements are conserved among other activating sulfotransferases. Our structural analysis reveals a unique substrate-binding pocket that accommodates bulky, complex substrates, suggesting a specialized adaptation for Sungeidine synthesis. Moreover, we have constructed a substrate docking model that provides insights into the molecular interactions between SgdX2 and Sungeidine F, enhancing our understanding of the enzyme's specificity and catalytic mechanism. The model supports a general acid-base catalysis mechanism, akin to other sulfotransferases, and underscores the minor role of disordered regions in substrate recognition. This integrative study of crystallography and computational modeling advances our knowledge of microbial secondary metabolite biosynthesis and may facilitate the development of novel biotechnological applications.
Topics: Sulfotransferases; Crystallography, X-Ray; Models, Molecular; Bacterial Proteins; Protein Conformation; Substrate Specificity; Catalytic Domain
PubMed: 38621346
DOI: 10.1016/j.bbrc.2024.149891 -
American Journal of Physiology. Cell... Jun 2022Heparan sulfate is a widely expressed polysaccharide in the extracellular matrix and on the cell surface. 3--sulfated heparan sulfate represents only a small percentage... (Review)
Review
Heparan sulfate is a widely expressed polysaccharide in the extracellular matrix and on the cell surface. 3--sulfated heparan sulfate represents only a small percentage of heparan sulfate from biological sources. However, this subpopulation is closely associated with biological functions of heparan sulfate. The 3--sulfated heparan sulfate is biosynthesized by heparan sulfate 3--sulfotransferase, which exists in seven different isoforms. This review article summarizes the recent progress in the substrate specificity studies of different 3--sulfotransferase isoforms involving the use of homogeneous oligosaccharide substrates and crystal structural analysis. The article also reviews a newly developed liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method to analyze the level of 3--sulfated heparan sulfate with high sensitivity and quantitative capability. This newly emerged technology will provide new tools to study the structure and function relationship of heparan sulfate.
Topics: Chromatography, Liquid; Heparitin Sulfate; Protein Isoforms; Sulfates; Sulfotransferases; Tandem Mass Spectrometry
PubMed: 35417268
DOI: 10.1152/ajpcell.00110.2022 -
Molecular and Cellular Endocrinology Apr 2013Estrogen sulfotransferase (SULT1E1) is a phase II drug-metabolizing enzyme known to catalyze sulfoconjugation of estrogens. 17β-estradiol (E2) plays a pivotal role in...
Estrogen sulfotransferase (SULT1E1) is a phase II drug-metabolizing enzyme known to catalyze sulfoconjugation of estrogens. 17β-estradiol (E2) plays a pivotal role in attenuating endothelial dysfunction. E2 can be further sulfated to estradiol sulfate (E2S) using SULT1E1. To date, there are no reports of expression and function of SULT1E1 in the endothelium. We identified that SULT1E1 is highly expressed in human umbilical vein endothelial cells (HUVECs) using immunofluorescence microscopy and Western immunoblot analyses. A synthesized siRNA targeting SULT1E1 was used to successfully suppress SULT1E1 expression and inhibit estrogen sulfation in HUVECs. This led to functional depletion, as confirmed by a SULT1E1 enzyme activity assay in vitro and by an in vivo estrogen sulfation assay. Knock-down of SULT1E1 in HUVECs resulted in regulation of genes involved in inflammation and lipid metabolism. Interestingly, this regulation was attenuated by PPARγ siRNA and by exposure to the PPARγ antagonist GW9662. Compared with cell response in the absence of estrogen, the effects of SULT1E1 interference on the inflammatory response and lipid metabolism related genes in the presence of 80nM estrogen were completely opposite. When exogenous estrogen was applied, cell responses depended on the ratio of E2 to E2S, due to the activity of SULT1E1, and the different regulation of these processes. It is suggested that E2 sulfation catalyzed by SULT1E1 plays an important role in modulating endothelial cell function.
Topics: Endothelial Cells; Estradiol; Human Umbilical Vein Endothelial Cells; Humans; Lipid Metabolism; Metabolic Detoxication, Phase II; PPAR gamma; RNA Interference; Sulfotransferases
PubMed: 23384540
DOI: 10.1016/j.mce.2013.01.020 -
Structural basis for the broad substrate specificity of the human tyrosylprotein sulfotransferase-1.Scientific Reports Aug 2017Tyrosylprotein sulfotransferases (TPSTs) are enzymes that catalyze post-translational tyrosine sulfation of proteins. In humans, there are only two TPST isoforms,...
Tyrosylprotein sulfotransferases (TPSTs) are enzymes that catalyze post-translational tyrosine sulfation of proteins. In humans, there are only two TPST isoforms, designated TPST1 and TPST2. In a previous study, we reported the crystal structure of TPST2, which revealed the catalytic mechanism of the tyrosine sulfation reaction. However, detailed molecular mechanisms underlying how TPSTs catalyse a variety of substrate proteins with different efficiencies and how TPSTs catalyze the sulfation of multiple tyrosine residues in a substrate protein remain unresolved. Here, we report two crystal structures of the human TPST1 complexed with two substrate peptides that are catalysed by human TPST1 with significantly different efficiencies. The distinct binding modes found in the two complexes provide insight into the sulfation mechanism for these substrates. The present study provides valuable information describing the molecular mechanism of post-translational protein modifications catalysed by TPSTs.
Topics: Binding Sites; Humans; Male; Models, Molecular; Molecular Structure; Peptides; Protein Binding; Protein Conformation; Protein Multimerization; Structure-Activity Relationship; Substrate Specificity; Sulfotransferases; Tyrosine
PubMed: 28821720
DOI: 10.1038/s41598-017-07141-8 -
International Journal of Biological... 2005Tyrosylprotein sulfotransferase (TPST), the enzyme responsible for the sulfation of tyrosine residues, has been identified and characterized in submandibular salivary...
Tyrosylprotein sulfotransferase (TPST), the enzyme responsible for the sulfation of tyrosine residues, has been identified and characterized in submandibular salivary glands previously (William et al. Arch Biochem Biophys 338: 90-96). Tyrosylprotein sulfotransferase catalyses the sulfation of a variety of secretory and membrane proteins and is believed to be present only in the cell. In the present study, this enzyme was identified for the first time in human saliva. Analysis of human saliva and parotid saliva for the presence of tyrosylprotein sulfotransferase revealed tyrosine sulfating activity displayed by both whole saliva and parotid saliva at pH optimum of 6.8. In contrast to tyrosylprotein sulfotransferase isolated from submandibular salivary glands, salivary enzyme does not require the presence of Triton X-100, NaF and 5'AMP for maximal activity. Similar to the submandibular TPST, the enzyme from saliva also required MnCl(2) for its activity. Maximum TPST activity was observed at 20 mM MnCl(2). The enzyme from saliva was immunoprecipitated and purified by immunoaffinity column using anti-TPST antibody. Affinity purified salivary TPST showed a single band of 50-54 kDa. This study is the first report characterizing a tyrosylprotein sulfotransferase in a secretory fluid.
Topics: Chlorides; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Humans; Immunoblotting; Kinetics; Manganese Compounds; Parotid Gland; Reference Values; Saliva; Sulfotransferases; Tyrosine
PubMed: 16244708
DOI: 10.7150/ijbs.1.141 -
The Journal of Biological Chemistry Nov 2001Dehydroepiandrosterone sulfotransferase (STD) is a hydroxysteroid sulfo-conjugating enzyme with preferential substrate specificity for C-19 androgenic steroids and C-24...
Dehydroepiandrosterone sulfotransferase (STD) is a hydroxysteroid sulfo-conjugating enzyme with preferential substrate specificity for C-19 androgenic steroids and C-24 bile acids. STD is primarily expressed in the liver, intestine and adrenal cortex. Earlier studies have shown that androgens inhibit the rat Std promoter function through a negative androgen response region located between -235 and -310 base pair positions (Song, C. S., Jung, M. H., Kim, S. C., Hassan, T., Roy, A. K., and Chatterjee, B. (1998) J. Biol. Chem. 273, 21856-21866). Here we report that the primary bile acid chenodeoxycholic acid (CDCA) also acts as an important regulator of the Std gene promoter. CDCA is a potent inducer of the Std gene, and its inducing effect is mediated through the bile acid-activated farnesoid X receptor (FXR), a recently characterized member of the nuclear receptor superfamily. The ligand-activated FXR acts as a heterodimer with the 9-cis-retinoic acid receptor (RXR) and regulates the Std gene by binding to an upstream region at base pair positions -169 to -193. This specific binding region was initially identified by bile acid responsiveness of the progressively deleted forms of the Std promoter in transfected HepG2 hepatoma and enterocyte-like Caco-2 cells. Subsequently, the precise RXR/FXR binding position was established by protein-DNA interaction using in vitro footprinting and electrophoretic mobility shift analyses. Unlike all other previously characterized FXR target genes, which contain an inverted repeat (IR) of the consensus hexanucleotide half-site (A/G)G(G/T)TCA with a single nucleotide spacer (IR-1), the bile acid response element of the Std promoter does not contain any spacer between the two hexanucleotide repeats (IR-0). A promoter-reporter construct carrying three tandem copies of the IR-0 containing -169/-193 element, linked to a minimal thymidine kinase promoter, can be stimulated more than 70-fold in transfected Caco-2 cells upon CDCA treatment. Autoregulation of the STD gene by its bile acid substrate may provide an important contributing role in the enterohepatic bile acid metabolism and cholesterol homeostasis.
Topics: Bile Acids and Salts; Binding Sites; DNA-Binding Proteins; Dimerization; Gene Expression Regulation, Enzymologic; Promoter Regions, Genetic; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoid X Receptors; Sulfotransferases; Transcription Factors; Transcriptional Activation
PubMed: 11533040
DOI: 10.1074/jbc.M107557200